The presentation of antigenic peptides by class I major histocompatibility complex (MHC) molecules plays a central role in the cellular immune response, since immune surveillance for detection of viral infections or malignant transformations is achieved by cytotoxic T lymphocytes (CTL), which inspect peptides bound to class I molecules on the surface of most cells (Yang et al., 1996). CTL eliminate infected cells by recognizing foreign antigens (Rock and Goldberg, 1999), which are processed in a proteasome-dependent manner and are presented by the MHC class I molecules. The multisubunit proteasomes, which degrade cytoplasmic proteins in an ATP and ubiquitin-dependent manner, are required for the generation of the antigenic peptides. Biochemical studies with proteasome inhibitors (Rock et al., 1994; Vinitsky et al., 1997) have provided evidence that the proteasome is responsible for the generation of class I-binding peptides. Indeed, in vitro enzymatic studies of isolated proteasomes have demonstrated that altered molecular organization of the proteasome induced by IFN is responsible for functional changes in the catalytic activity. This ultimately results in changes to antigen processing (Driscoll et al., 1993; Fruh and Yang, 1999; Gaczynska et al., 1996; Gaczynska et al., 1993; Gaczynska et al., 1994). Moreover, in vivo evidence obtained from analysis of LMP2 or LMP7 knock-out mice (Fehling et al., 1994; Van Kaer et al., 1994) indicates that IFN-induced proteasome subunits play a major role in proteasome-mediated antigen processing. Both LMP2 and LMP7 knock-out mice are deficient in the generation of a subset of antigenic peptides. Thus, the proteasome subunit exchange is a fundamental mechanism for modulating proteasome activities by cytokines during immune responses (Fruh et al., 1997).
In eukaryotes, proteasome activities are modulated by specific regulatory proteins that form complexes with proteasomes (Yang et al., 1996). Two regulatory complexes, the ATPase complex and PA28, have been studied to some extent. The ATPase complex associates with the 20 S proteasome in an ATP-dependent manner, resulting in the 26 S proteasome (Rock and Goldberg, 1999). This 26 S proteasome is involved in the degradation of protein substrates in an ubiquitin-dependent manner (Rock and Goldberg, 1999). The proteasome regulator PA28 has been shown to associate with the 20 S proteasome in vitro (Chu-Ping et al., 1992; Chu-Ping et al., 1993) and in vivo (Yang et al., 1995) in an ATP-independent manner. Association of these regulatory complexes appears to be reversible and regulated by phosphorylation (Yang et al., 1995). It is conceivable that evolutionary divergence of these regulatory complexes is coupled with their functional specialization and that regulatory mechanisms exist that render antigenic peptides more likely to become available to class I molecules during immune responses. Antigen degradation could occur in two steps, namely, initial degradation of whole antigen into intermediate sized fragments by 26 S proteasomal complexes followed by degradation of these fragments by the PA28/20 S proteasomal complexes to produce peptides of 8-10 residues in length (Fourie and Yang, 1998; Fruh and Yang, 1999). Indeed, in vitro kinetic studies on the influence of PA28 on peptide cleavage and specificity of the proteasome (Chu-Ping et al., 1992) indicate that PA28 changes the cleavage behavior of the proteasome in a characteristic qualitative and quantitative manner. In the absence of PA28, the proteasome digests substrates by consecutive and independent single cleavages. Upon association with PA28, products generated by two flanking cleavages appear immediately as main products, while the generation of single-cleavage products is strongly reduced (Dick et al., 1996). Since this PA28-induced, coordinated double-cleavage mechanism appears to optimize the generation of dominant T-cell epitopes (Groettrup et al., 1996), the regulation of PA28 expression by IFN plays an essential role in proteasome-mediated antigen processing (Ahn et al., 1996; Fruh and Yang, 1999).
The peptidase activities of the proteasome can be activated in vitro by the proteasome regulator PA28.alpha., .beta., or both (Realini et al., 1997; Song et al., 1997). In mice, there are at least two functional copies for PA28.alpha., while PA28.beta. has only one functional copy (Li et al., 1998). PA28.alpha. itself is capable of forming homoheptamers in vitro (Johnston et al., 1997; Knowlton et al., 1997). An in vivo role for PA28 remains unknown, although PA28 has been implicated in playing a role in MHC class I antigen presentation (Dick et al., 1996; Groettrup et al., 1996). The underlying mechanism by which PA28 modulates proteasome function in antigen processing remains elusive. More specifically the individual roles for PA28.alpha. and PA28.beta. in vivo on immuno-proteasomes and their relationship to each other have yet to be understood. Understanding the roles of these proteins, and specifically PA28.beta., should aid in understanding therapeutically important disease states including auto-immunity, transplantation, inflammation, and cancer immunology. Greater understanding of the roles that proteasome-dependent antigen presentation for these conditions is expected to open new mechanisms of therapeutic intervention or modulation.
The present invention provides a means to dissect the functional role of PA28 in different cell types, such as cytotoxic T cells and antigen presenting cells. The PA28.beta. gene was disrupted by homologous recombination and PA28 deficient cells were prepared. The in vivo effect of deficient proteasome-dependent major histocompatibility complex (MHC) classes I antigen processing is analyzed in PA28-deficient transgenic animals.